Effect of the At-CDC27a gene on Nicotiana benthamiana phenotype and accumulation of recombinant proteins.

In this study the anaphase promoting complex subunit CDC27a from Arabidopsis thaliana was introduced in the genome of Nicotiana benthamiana by Agrobacterium tumefaciens. The presence of the At-CDC27a gene facilitates plant biomass production. Compared to wild type N. benthamiana the leaf mass fraction of the best performing transgenic line At-CDC27a-29 was increased up to 154%. The positive effect of the At-CDC27a expression on leaf biomass accumulation was accompanied by an enlarged total leaf area. Furthermore, the ectopic expression of the At-CDC27a also affected cellular conditions for the production of foreign proteins delivered by the TRBO vector. In comparison to the non-transgenic control, the protein accumulation in the At-CDC27a-29 plant host increased up to 146% for GFP and up to 181% for scFv-TM43-E10. Collectively, the modified N. benthamiana plants developed in this study might be useful to improve the yield of recombinant proteins per biomass unit in closed facilities.

At-CycD2 Enhances Accumulation of Above-Ground Biomass and Recombinant Proteins in Transgenic Nicotiana benthamiana Plants.

Transient expression in Nicotiana benthamiana holds great potential for recombinant protein manufacturing due to its advantages in terms of speed and yield compared to stably transformed plants. To continue improving the quantity of recombinant proteins the plant host will need to be modified at both plant and cellular levels. In attempt to increase leaf mass fraction, we transformed N. benthamiana with the At-CycD2 gene, a positive regulator of the cell cycle. Phenotypic characterization of the T1 progeny plants revealed their accelerated above-ground biomass accumulation and enhanced rate of leaf initiation. In comparison to non-transgenic control the best performing line At-CycD2-15 provided 143 and 140% higher leaf and stem biomass fractions, respectively. The leaf area enlargement of the At-CycD2-15 genotype was associated with the increase of epidermal cell number compensated by slightly reduced cell size. The production capacity of the At-CycD2-15 transgenic line was superior to that of the non-transgenic N. benthamiana. The accumulation of transiently expressed GFP and scFv-TM43-E10 proteins per unit biomass was increased by 138.5 and 156.7%, respectively, compared to the wild type. With these results we demonstrate the potential of cell cycle regulator gene At-CycD2 to modulate both plant phenotype and intracellular environment of N. benthamiana for enhanced recombinant protein yield.

The carrot monoterpene synthase gene cluster on chromosome 4 harbours genes encoding flavour-associated sabinene synthases.

In plants, low molecular weight terpenes produced by terpene synthases (TPS) contribute to multiple ecologically and economically important traits. The present study investigates a carrot terpene synthase gene cluster on chromosome 4 associated with volatile monoterpene production. Two carrot mutants, yellow and cola, which are contrasting in the content of low molecular weight terpenes, were crossed to develop an F2 mapping population. The mapping analysis revealed overlapping QTLs on chromosome 4 for sabinene, α-thujene, α-terpinene, γ-terpinene, terpinen-4-ol and 4-carene. The genomic region of this locus includes a cluster of five terpene synthase genes (DcTPS04, DcTPS26, DcTPS27, DcTPS54 and DcTPS55). DcTPS04 and DcTPS54 displayed genotype- and tissue-specific variation in gene expression. Based on the QTL mapping results and the gene expression patterns, DcTPS04 and DcTPS54 were selected for functional characterization. In vitro enzyme assays showed that DcTPS54 is a single-product enzyme catalysing the formation of sabinene, whereas DcTPS04 is a multiple-product terpene synthase producing α-terpineol as a major product and four additional products including sabinene, ß-limonene, ß-pinene and myrcene. Furthermore, we developed a functional molecular marker that could discriminate carrot genotypes with different sabinene content in a set of 85 accessions.

Enhanced foreign protein accumulation in Nicotiana benthamiana leaves co-infiltrated with a TMV vector and plant cell cycle regulator genes.

In this short communication, we report that the cell cycle checkpoint genes At-CycD2 and At-CDC27a from Arabidopsis thaliana enhance the transient heterologous protein expression in Nicotiana benthamiana. We selected a well-studied and widely used virus expression vector based on TMV for the delivery of recombinant proteins into the host plant. Co-infiltration of TMV-gfp and binary expression vectors carrying the At-CycD2 and At-CDC27a genes, respectively, resulted in enhanced GFP fluorescence in agroinoculated leaves. These findings corresponded with the observation of (1) higher mRNA levels for TMV and gfp and (2) increased GFP protein accumulation. Furthermore, by co-delivery of the TMV-scFv-TM43-E10 and At-CycD2/At-CDC27a expressing constructs we observed an enhanced amount of the scFv-TM43-E10 antibody fragment compared to the delivery of the TMV-scFv-TM43-E10 alone. We anticipate that this finding might be adapted for enhancing foreign protein production in N. benthamiana as the host plant.

Transient plant production of Salmonella Typhimurium diagnostic antibodies.

Salmonella Typhimurium is one of the most important zoonotic pathogens worldwide and a major cause of economic losses in the pig production chain. The emergence of multi-drug resistant strains over the past years has led to considerations about an enhanced surveillance of bacterial food contamination. Currently, ELISA is the method of choice for high throughput identification of S. Typhimurium. The sensitivity and specificity of this assay might be improved by application of new diagnostic antibodies. We focused on plant-based expression of candidate diagnostic TM43-E10 antibodies discovered using as antigen the S. Typhimurium OmpD protein. The scFv-TM43-E10 and scFv-Fc-TM43-E10 antibody derivatives have been successfully produced in N. benthamiana using a deconstructed movement-deficient PVX vector supplemented with the γb silencing suppressor from Poa semilatent virus. The plant-made antibodies showed the same antigen-binding specificity as that of the microbial/mammalian cell-produced counterparts and could recognize the OmpD antigen in S. Typhimurium infected plant samples.

Transient Production of Recombinant Pharmaceutical Proteins in Plants: Evolution and Perspectives.

During the last two decades, the production of pharmaceutical proteins in plants evolved from proof of concept to established technology adopted by several biotechnological companies. This progress is particularly based on intensive research starting stable genetic transformation and moving to transient expression. Due to its advantages in yield and speed of protein production transient expression platforms became the leading plant-based manufacturing technology. Current transient expression methods rely on Agrobacteriummediated delivery of expression vectors into plant cells. In recent years, great advances have been made in the improvement of expression vectors, host cell engineering as well as in the development of commercial manufacturing processes. Several GMP-certified large-scale production facilities exist around the world to utilize agroinfiltration method. A number of pharmaceutical proteins produced by transient expression are currently in clinical development. The great potential of transient expression platform in respect to rapid response to emerging pandemics was demonstrated by the production of experimental ZMapp antibodies against Ebola virus as well as influenza vaccines. This review is focused on current design, status and future perspectives of plant transient expression system for the production of biopharmaceutical proteins.

Marker Removal in Transgenic Plants Using Cre Recombinase Delivered with Potato Virus X.

In this chapter we present an alternative method to develop marker-free transgenic plants. It makes use of the Cre/loxP recombination system from bacteriophage P1 and consists of two essential components. The first component is the transgenic plant containing a loxP-flanked marker gene. The second component is a cre transient expression vector based on potato virus X. The great benefit of this transient delivery method consists in the avoidance of stable integration of the cre recombinase gene into the plant genome. Upon infection of the loxP-target plant with PVX-Cre, the virus spreads systemically through the plant and causes the recombinase-mediated excision of the marker gene. Marker-free transgenic loci can be transmitted to the progeny by plant regeneration from PVX-Cre systemically infected leaves or self-pollination of virus-infected plants. The protocol covers generation of loxP-target transgenic plants, PVX-mediated delivery of Cre recombinase protein, phenotypic and molecular analysis of recombination events, and transmission of marker-free transgenic loci to the next generation. The transient expression system described in this chapter can be adapted for marker gene removal in other plant species that are amenable for virus infection.

Plant 4/1 protein: potential player in intracellular, cell-to-cell and long-distance signaling.

Originally isolated as a result of its ability to interact with the movement protein of Tomato spotted wilt virus in a yeast two-hybrid system, the 4/1 protein is proving to be an excellent tool for studying intracellular protein trafficking and intercellular communication. Expression of 4/1 in vivo is tightly regulated, first appearing in the veins of the cotyledon and later in the vasculature of the leaf and stem in association with the xylem parenchyma and phloem parenchyma. Structural studies indicate that 4/1 proteins contain as many as five coiled-coil (CC) domains; indeed, the highest level of sequence identity among 4/1 proteins involves their C-terminal CC domains, suggesting that protein-protein interaction is important for biological function. Recent data predict that the tertiary structure of this C-terminal CC domain is strikingly similar to that of yeast protein She2p; furthermore, like She2p, 4/1 protein exhibits RNA-binding activity, and mutational analysis has shown that the C-terminal CC domain is responsible for RNA binding. The 4/1 protein contains a nuclear export signal. Additional microscopy studies involving leptomycin and computer prediction suggest the presence of a nuclear localization signal as well.

Possible role of the Nt-4/1 protein in macromolecular transport in vascular tissue.

The Arabidopsis thaliana 4/1 (At-4/1) protein has a highly α-helical structure with potential to interact both with itself and other protein ligands, including the movement proteins of some plant viruses; the Nicotiana tabacum ortholog (Nt-4/1) has similar structure. Here we describe localization of GUS expression in transgenic N. tabacum seedlings under control of the Nt-4/1 promoter, which indicates that transcription is associated with the veins at certain developmental stages, and especially in the hypocotyl. Viroid accumulation and movement was altered in plants in which 4/1 expression was reduced by virus-induced gene silencing. These localization studies support a role of 4/1 in signaling in the vasculature,including mobility of pathogen-related and cellular RNAs.

A developmentally regulated Cre-lox system to generate marker-free transgenic Brassica napus plants.

In this chapter, a strategy for engineering marker-free Brassica napus plants is described. It is based on the Cre-lox site-specific recombination system and includes three essential steps. At first, the binary vector pLH-nap-lx-cre-35S-bar-lx-vst has been designed. In this vector, the cre gene and the bar expression cassette are flanked by two lox sites in direct orientation. The lox-flanked sequence is placed between a seed-specific napin promoter and a coding region for the vstI gene. At the second step, the cre-bar vector was transferred into B. napus hypocotyl explants by Agrobacterium tumefaciens-mediated transformation. Finally, T1 progeny was tested for excision of the marker gene at phenotypic and molecular levels. PCR, sequencing, and Southern blot analysis confirmed complete and precise deletion of the lox-flanked DNA region. This developmentally regulated Cre-lox system can be applied to remove undesirable DNA in transgenic plants propagated by seeds.

Utilization of PVX-Cre expression vector in potato.

Trait genes are usually introduced into the plant genome together with a marker gene. The last one becomes unnecessary after transgene selection and characterization. One of the strategies to produce transgenic plants free from the selectable marker is based on site-specific recombination. The present study employed the transient Cre-lox system to remove the nptII marker gene from potato. Transient marker gene excision involves introduction of Cre protein in lox-target plants by PVX virus vector followed by plant regeneration. Using optimized experimental conditions, such as particle bombardment infection method and application of P19 silencing suppressor protein, 20-27% of regenerated plants were identified by PCR analysis as marker-free. Based on our comparison of the recombination frequencies observed in this study to the efficiency of other methods to avoid or eliminate marker genes in potato, we suggest that PVX-Cre mediated site-specific excisional recombination is a useful tool to generate potato plants without superfluous transgenic sequences.

Orthologues of a plant-specific At-4/1 gene in the genus Nicotiana and the structural properties of bacterially expressed 4/1 protein.

Arabidopsis thaliana At-4/1 is the protein of unknown function capable of polar localization in plant cells and intercellular trafficking. In this work, we cloned cDNAs and chromosomal genes of At-4/1 orthologues from several Nicotiana species. Similarly to the 4/1 genes of A. thaliana and Oryza sativa, Nicotiana 4/1 genes have eight exons and seven introns but are considerably longer due to their larger introns. The allotetraploid genome of Nicotiana tabacum, which is known to consist of the 'S genome' originated from Nicotiana sylvestris and the 'T genome' derived from Nicotiana tomentosiformis, encodes two 4/1 genes. The T genome-encoded 4/1 gene, but not that of the S genome, contains a SINE-like transposable element in its intron 2. The 4/1 genes of Nicotiana hesperis and Nicotiana benthamiana lack such an element in the intron 2, but possess a related SINE-like sequence in their intron 4. Collectively, the sequence analysis data provide an insight into the organization of 4/1 genes in flowering plants and the patterns of evolution in the genus Nicotiana. The Nicotiana 4/1 proteins and those of other flowering plants show a significant level of sequence similarity. Computer-assisted analysis was further used to compare their predicted secondary structures. Several algorithms confidently predicted the presence of several coiled-coil domains occupying similar positions in different 4/1 proteins. Analysis of circular dichroism spectra carried out for bacterially expressed N. tabacum 4/1 protein (Nt-4/1) and its N- and C-terminally truncated mutants confirmed that the secondary structure of Nt-4/1 is generally alpha-helical. The C-terminal region of Nt-4/1 was found to undergo a partial proteolysis in Escherichia coli cells. Differential scanning calorimetry of Nt-4/1 protein and its mutants revealed three calorimetric domains most probably corresponding to the N-terminal, central, and C-terminal structural domains of the protein.

Tuber-specific cphA expression to enhance cyanophycin production in potatoes.

The production of biodegradable polymers that can be used to substitute petrochemical compounds in commercial products in transgenic plants is an important challenge for plant biotechnology. Nevertheless, it is often accompanied by reduced plant fitness. To decrease the phenotypic abnormalities of the sprout and to increase polymer production, we restricted cyanophycin accumulation to the potato tubers by using the cyanophycin synthetase gene (cphA(Te)) from Thermosynechococcus elongatus BP-1, which is under the control of the tuber-specific class 1 promoter (B33). Tuber-specific cytosolic (pB33-cphA(Te)) as well as tuber-specific plastidic (pB33-PsbY-cphA(Te)) expression resulted in significant polymer accumulation solely in the tubers. In plants transformed with pB33-cphA(Te), both cyanophycin synthetase and cyanophycin were detected in the cytoplasm leading to an increase up to 2.3% cyanophycin of dry weight and resulting in small and deformed tubers. In B33-PsbY-cphA(Te) tubers, cyanophycin synthetase and cyanophycin were exclusively found in amyloplasts leading to a cyanophycin accumulation up to 7.5% of dry weight. These tubers were normal in size, some clones showed reduced tuber yield and sometimes exhibited brown sunken staining starting at tubers navel. During a storage period over of 32 weeks of one selected clone, the cyanophycin content was stable in B33-PsbY-cphA(Te) tubers but the stress symptoms increased. However, all tubers were able to germinate. Nitrogen fertilization in the greenhouse led not to an increased cyanophycin yield, slightly reduced protein content, decreased starch content, and changes in the amounts of bound and free arginine and aspartate, as compared with control tubers were observed.

Developmentally regulated site-specific marker gene excision in transgenic B. napus plants.

We have developed a self-excision Cre-vector to remove marker genes from Brassica napus. In this vector cre recombinase gene and bar expression cassette were inserted between two lox sites in direct orientation. These lox-flanked sequences were placed between the seed-specific napin promoter and the gene of interest (vstI). Tissue-specific cre activation resulted in simultaneous excision of the recombinase and marker genes. The vector was introduced into B. napus by Agrobacterium-mediated transformation. F1 progeny of seven lines with single and multiple transgene insertions was subjected to segregation and molecular analysis. Marker-free plants could be detected and confirmed by PCR and Southern blot in all transgenic lines tested. The recombination efficiency expressed as a ratio of plants with complete gene excision to the total number of investigated plants varied from 13 to 81% dependent on the transgene copy number. Potential application of this system would be the establishment of marker-free transgenic plants in generatively propagated species.

Agroinfiltration as a tool for transient expression of cre recombinase in vivo.

Agroinfiltration was used to express transiently cre recombinase from bacteriophage P1 in planta. Activation of gfp expression after cre-mediated excision of a bar intervening sequence served as a marker to monitor site-specific recombination events in lox-target N. benthamiana plants. Gfp expressing regenerants from A. tumefaciens infiltrated leaves were obtained with an efficiency of about 34%. In 20% of the regenerants bar gene excision was due to the expression of stably integrated cre gene, whereas in 14% of plants site-specific recombination was a consequence of transient cre expression. Phenotypic and molecular data indicated that the recombined state has been transferred to the T(1 )generation. These results demonstrate the suitability of agroinfiltration for the expression of cre recombinase in vivo.